A variety of techniques as described in the Methods of Enzymology, Vols. 70-73, Ed. J. J. Langone and H. U. Unhais, Academic Press, 1981, are routinely used to assay fluids for molecules present in micromolar amounts or less, that are indicative of disease, or alterations in the normal physiology of a living system. Nearly all of these techniques are premised on the use of molecules that specifically recognize the molecule sought to be assayed, the former being termed a ligand-recognition molecule, and the latter a ligand. Perhaps the best example is the detection of antigen with antibody, although, as described by Erb, L. et al in Steroids Vol. 39, p. 33 (1981) other nonimmune related specific binding receptors have been used. Generally such assays involve the formation of a complex, either in solution, or on a solid surface, of a labelled ligand bound to a ligand-recognition molecule. The amount of ligand in the fluid sought to be assayed is determined by its ability to either compete off ligand from the ligand-recognition molecule subsequent to complex formation, or to compete with ligand for binding to the ligand-recognition molecule simultaneous to complex formation. Since the resulting amount of labelled ligand bound to the ligand-recognition molecule is inversely related to the amount of ligand present in the fluid, the presence of the latter can be ascertained and/or quantitated. A second method of detecting ligands is to label the ligand-recognition molecule such that the amount of ligand-recognition molecule detected reflects the amount of complex formed which is in turn a function of ligand concentration present in the fluid being assayed.
Both ligands or ligand-recognition molecules are commonly labelled either by direct covalent attachment of radioactive atoms, such as 125.sub.I, to the molecule, or by synthesis of the ligand or ligand-recognition molecule using radioactive starting materials so as to incorporate radioactive atoms into their structure. In lieu of using radio labels, ligands or ligand-recognition molecules can be labelled by covalent attachment of either fluorescent or enzymatic molecules. The latter, when incubated with the appropriate substrate give readily detectable color reactions, and is termed ELISA (Enzyme Linked Immunosorbant Assay).
The presently used immunoassays are sensitive and versatile enough to detect most ligands. Nonetheless, these assays have some undesirable features. Notably they rely on the use and detection of radioactive substances which necessarily require expensive instrumentation, generally scintillation or gamma counters, in either a medical or research setting. Also, because of the heightened awareness of the dangers associated with radiation, it is becoming increasingly difficult, partly due to newly implemented state regulations, to dispose of such substances after they have been used. Thus, other less expensive methods of performing immunochemical assays that do not use radioactive labels are desirable. To some extent the detection of ligands or ligand-recognition molecules based on enzyme labels have circumvented the problems associated with the use of radioactive tracers. Nevertheless, these assays have certain limitations, the major one being the large size of the enzymes used which, when bound to the ligand or ligand-recognition molecule, have the potential to alter their normal structure and hence mask their recognition sites.